We aim to develop a method for virus discovery that is independent of either amplification or purification of viral particles. RNA interference (RNAi) functions as an antiviral immunity in fungi, plants, fruitflies, mosquitoes and nematodes. In this immunity, dsRNA produced during replication of viral RNA genomes or convergent transcription of viral DNA genomes is processed into 21- to 24-nucleotide virus-derived small interfering RNAs (siRNAs) by Dicer to guide specific viral RNA clearance. We have recently cloned and sequenced by the next generation sequencing technologies viral siRNAs produced in plant and fruitfly cells infected with positive-strand RNA viruses. Our results show that viral siRNAs produced by the host immune system in response to viral infection are overlapping in sequence and can be assembled back into long continuous fragments (contigs) of the infecting viral RNA genome using available computer programs. Thus, we hypothesize that deep sequencing and assembly of virus-derived siRNAs can be employed as a new approach for virus discovery. This hypothesis is supported by discovering three new viruses after examination of a recently small RNA population of a Drosophila S2 cell line sequenced by the Illumina platform. Thus, Aim 1 will firstly develop the pipeline for discovering new arthropod viruses by constructing and sequencing 12 small RNA libraries from Drosophila cell lines collected around the U.S. Aim 2 will construct and sequence 6 small RNA libraries from mosquito cell lines and 45 small RNA libraries from adult female mosquitoes collected from the U.S. and China by our collaborators. Many important human and animal viruses are transmitted by mosquitoes and many human diseases have no identified etiology. Therefore, we expect that this project will establish an exciting approach for virus discovery, facilitate identification of etiology of human diseases, and increase our understanding of the virus diversity and evolution of arthropod and arthropod-borne viruses. This project aims to develop a completely new approach for virus discovery in mosquitoes. Many important human and animal viruses are transmitted by mosquitoes and many human diseases have no identified etiology. Therefore, it is likely that this project will facilitate identification of etiology of human diseases and increase our understanding of the virus diversity and evolution of arthropod and arthropod-borne viruses.